Antibodies that bind peptides from the hPTH sequence (1-37)

ABSTRACT

The present invention is directed to peptides from the sequence of hPTH(1-37), which contain α-helical amino acid sequence regions and/or non-structured amino acid sequence regions, said peptides being capable of inducing antibodies when injected into animals. Furthermore, the invention is directed to a diagnostic agent and antibodies obtainable by immunizing animals using the peptides according to the invention.

This application was filed under 35 U.S.C. § 371 and claims priorityfrom PCT/EP95/03757, filed Sep. 22, 1995.

The present invention relates to peptides from the sequence ofhPTH(1-37), and the use of said peptides in the preparation of an agentfor diagnosing biologically active hPTH.

Human parathyroid hormone (hPTH), a linear polypeptide having 84 aminoacids, plays an important role in the regulation of the calciummetabolism. The metabolism of this hormone gives rise to a large numberof C-terminal fragments, the biological functions of which have not yetbeen elucidated. The hPTH(1-37) has been established as a circulatingN-terminal fragment (EP-A 0 349 545). This fragment has the fullbiological activity of the entire hormone. However, upon loss of thefirst amino acid, serine, the activity significantly decreases and islost completely without the first two amino acids, serine and valine.

Serum levels in the range of 10⁻¹² mol/l are measured for the intacthormone hPTH(1-84) and for the N-terminal fragment. Immunologicalmeasuring procedures are employed to determine such low concentrations.Here, the most valid results are provided by measuring proceduresaccording to the double antibody or sandwich principle (e.g., thetwo-site radioimmunometric assay, IRMA, or the sandwich enzyme-linkedimmuno sorbent assay, Sandwich ELISA). For hPTH(1-84), such assays arecommercially available. For the measurement of hPTH(1-34), an assayaccording to the double antibody principle is not known.

Here, two antibodies are required. In order to avoid mutual sterichindrance, they must be capable of recognizing antigen epitopes locatedat a sufficient distance from each other. When immunizing using theintact antigen, a heterogeneous mixture of various antibodies isobtained, which first must be subjected to an expensive purification inorder to conduct a sandwich assay. According to theoretical calculationsby B. A. Jameson and H. Wolf, The Antigenic Index: A Novel Algorithm forPredicting Antigenic Determinants, CABIOS 4, p. 181-186, 1988; it hasbeen possible so far to detect a preferred sequence having immunogenicactivity in the region of the amino acids 7-14 at the N-terminus.Immunization with N-terminal fragments according to established methodspredominantly results in antibodies which, as has been described forhPTH(1-34) (J. Tampe, P. Brozio, H. E. Manneck, A. Miβbichler, E. Blind,K. B. Millers, H. SchmidtGayk, and F. P. Armbruster, Characterisation ofAntibodies Against Human N-Terminal Parathyroid Hormone by EpitopeMapping; J. Immunoassay 13, p. 1-13, 1992), bind in the region of theseamino acids. However, these antibodies are not capable of discriminatingbetween biologically active and biologically inactive PTH(1-84) orfragments thereof lacking the first two amino acids serine and valine.

The technical problem which this invention is based upon is to providepeptides by means of which it is possible to eliminate theabove-mentioned drawbacks in the diagnosis of biologically active hPTH.

Surprisingly, the technical problem described above is solved by meansof the following peptides from the sequence of hPTH(1-37):

    hPTH 1-10 SEQ I.D. NO.1                                                       NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6     -Leu.sup.7 -Met.sup.8 -His.sup.9 -Asn.sup.10 -OH                                                                      (1)                                  hPTH 1-9 SEQ I.D. NO.2                                                        NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6     -Leu.sup.7 -Met.sup.8 -His.sup.9 -OH    (2)                                  hPTH 1-8 SEQ I.D. NO.3                                                        NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6     -Leu.sup.7 -Met.sup.8 -OH               (3)                                  hPTH 1-7 SEQ I.D. NO.4                                                        NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6     -Leu.sup.7 -OH                          (4)                                  hPTH 1-6 SEQ I.D. NO.5                                                        NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -Gln.sup.6     -OH                                     (5)                                  hPTH 1-5 SEQ I.D. NO.6                                                        NH.sub.2 -Ser.sup.1 -Val.sup.2 -Ser.sup.3 -Glu.sup.4 -Ile.sup.5 -OH                                                    (6)                                  hPTH 9-18 SEQ I.D. NO.7                                                       NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13           -His.sup.14 -Leu.sup.15 -Asn.sup.16 -Ser.sup.17 -Met.sup.18 -OH                                                        (7)                                  hPTH 10-18 SEQ I.D. NO.8                                                      NH.sub.2 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13 -His.sup.14          -Leu.sup.15 -Asn.sup.16 -Ser.sup.17 -Met.sup.18 -OH                                                                    (8)                                  hPTH 11-18 SEQ I.D. NO.9                                                      NH.sub.2 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13 -His.sup.14 -Leu.sup.15          -Asn.sup.16 -Ser.sup.17 -Met.sup.18 -OH  (9)                                  hPTH 12-18 SEQ I.D. NO.10                                                     NH.sub.2 -Gly.sup.12 -Lys.sup.13 -His.sup.14 -Leu.sup.15 -Asn.sup.16          -Ser.sup.17 -Met.sup.18 -OH              (10)                                 hPTH 13-18 SEQ I.D. NO.11                                                     NH.sub.2 -Lys.sup.13 -His.sup.14 -Leu.sup.15 -Asn.sup.16 -Ser.sup.17          -Met.sup.18 -OH                          (11)                                 hPTH 14-18 SEQ I.D. NO.12                                                     NH.sub.2 -His.sup.14 -Leu.sup.15 -Asn.sup.16 -Ser.sup.17 -Met.sup.18                                                   (12)                                 hPTH 9-17 SEQ I.D. NO.13                                                      NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13           -His.sup.14 -Leu.sup.15 -Asn.sup.16 -Ser.sup.17 -OH                                                                    (13)                                 hPTH 9-16 SEQ I.D. NO.14                                                      NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13           -His.sup.14 -Leu.sup.15 -Asn.sup.16 -OH  (14)                                 hPTH 9-15 SEQ I.D. NO.15                                                      NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13           -His.sup.14 -Leu.sup.15 -OH              (15)                                 hPTH 9-14 SEQ I.D. NO.16                                                      NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13           -His.sup.14 -OH                          (16)                                 hPTH 9-13 SEQ I.D. NO.17                                                      NH.sub.2 -His.sup.9 -Asn.sup.10 -Leu.sup.11 -Gly.sup.12 -Lys.sup.13                                                    (17)                                 hPTH 24-37 SEQ I.D. NO.18                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -                 Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH                                                                     (18)                                 hPTH 25-37 SEQ I.D. NO.19                                                     NH.sub.2 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28 -Gln.sup.29          -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -                             Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH                                                                     (19)                                 hPTH 26-37 SEQ I.D. NO.20                                                     NH.sub.2 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28 -Gln.sup.29 -Asp.sup.30          -Val.sup.31 -His.sup.32 -Asn.sup.33 -                                         Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH                                                                     (20)                                 hPTH 27-37 SEQ I.D. NO.21                                                     NH.sub.2 -Lys.sup.27 -Leu.sup.28 -Gln.sup.29 -Asp.sup.30 -Val.sup.31          -His.sup.32 -Asn.sup.33 -                                                     Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH                                                                     (21)                                 hPTH 28-37 SEQ I.D. NO.22                                                     NH.sub.2 -Leu.sup.28 -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32          -Asn.sup.33 -Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH                                                        (22)                                 hPTH 29-37 SEQ I.D. NO.23                                                     NH.sub.2 -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33          -Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH                                                                    (23)                                 hPTH 30-37 SEQ I.D. NO.24                                                     NH.sub.2 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -Phe.sup.34          -Val.sup.35 -Ala.sup.36 -Leu.sup.37 -OH  (24)                                 hPTH 31-37 SEQ I.D. NO.25                                                     NH.sub.2 -Val.sup.31 -His.sup.32 -Asn.sup.33 -Phe.sup.34 -Val.sup.35          -Ala.sup.36 -Leu.sup.37 -OH              (25)                                 hPTH 32-37 SEQ I.D. NO.26                                                     NH.sub.2 -His.sup.32 -Asn.sup.33 -Phe.sup.34 -Val.sup.35 -Ala.sup.36          -Leu.sup.37 -OH                          (26)                                 hPTH 33-37 SEQ I.D. NO.27                                                     NH.sub.2 -Asn.sup.33 -Phe.sup.34 -Val.sup.35 -Ala.sup.36 -Leu.sup.37                                                   (27)                                 hPTH 24-36 SEQ I.D. NO.28                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -                 Phe.sup.34 -Val.sup.35 -Ala.sup.36 -OH   (28)                                 hPTH 24-35 SEQ I.D. NO.29                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -                 Phe.sup.34 -Val.sup.35 -OH               (29)                                 hPTH 24-34 SEQ I.D. NO.30                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -                 Phe.sup.34 -OH                           (30)                                 hPTH 24-33 SEQ I.D. NO.31                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -Asn.sup.33 -OH                                                        (31)                                 hPTH 24-32 SEQ I.D. NO.32                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -His.sup.32 -OH                                                                    (32)                                 hPTH 24-31 SEQ I.D. NO.33                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -Asp.sup.30 -Val.sup.31 -OH  (33)                                 hPTH 24-29 SEQ I.D. NO.34                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28          -Gln.sup.29 -OH                          (34)                                 hPTH 24-38 SEQ I.D. NO.35                                                     NH.sub.2 -Leu.sup.24 -Arg.sup.25 -Lys.sup.26 -Lys.sup.27 -Leu.sup.28                                                   (35)                             

The indicated sequences represent essential features of the secondarystructure in their primary structure, as can be demonstrated bysupporting NMR data. One precondition to this end was a determination ofthe PTH(1-37) secondary structure in physiological solution.

The above-mentioned regions of conspicuous structure have goodimmunogenic activity. Antibodies are formed, binding to the first aminoacids of the N-terminus. Deficiency of only two amino acids gives riseto a substantial loss in affinity. Because these amino acids areindispensable for the biological activity to arise, it is possible byusing the peptides of the invention to obtain antibodies recognizingonly hPTH and fragments thereof which are biologically active.

Furthermore, antibodies can be produced which detect the mid-region9-15, as well as antibodies giving C-terminal binding in the region ofthe amino acids 30-37. According to the invention, it is thereforepossible to produce antibodies against hPTH(1-37) regions which,according to theoretical calculations, do not exhibit immunogenicactivity within the entire molecule. In addition, these regions areseparated from each other by such a far distance that no sterichindrance is present which would prevent simultaneous binding of twoantibodies.

In preferred embodiments, the peptides may be modified at the N-terminalend, in the side-chain and/or at the C-terminal end, namely, taking theform of acetylation, amidation, phosphorylation and/or glycosylationproducts.

Eventually, the peptides of the invention may also be bound to carrierproteins such as hemocyanin, thyroglobulin, bovine serum albumin,ovalbumin, or mouse serum albumin etc. Binding to the carrier proteinsis preferably effected using carbodiimide or formaldehyde.

The peptides of the invention may be used in the preparation of adiagnostic agent. The diagnostic agent of the invention can be obtainedusing the per se known immunization of animals with at least one of thepeptides according to the invention. Following immunization, animmunoglobulin fraction can be isolated from the immunized animals,which contains antibody fractions having an antibody titer against atleast one of the peptides of the invention. The invention is alsodirected to the antibodies thus obtained. In addition to the completeantibodies consisting of F_(ab) and F_(c), fragments thereof such asF_(ab) or fragments of the antibodies being idiotypes of peptideepitopes may also be used in an alternative embodiment.

The peptides according to the invention are suitable for preparing anagent for the diagnosis of biologically active hPTH(1-37).

Referring to the following examples, the invention will be described inmore detail.

EXAMPLE 1

Solid-Phase Synthesis of Peptides

The method of the invention for synthesizing the peptides is based onthe peptide synthesis using a solid support. Each of the C-terminalamino acids is bound to the support material in the presence ofdicyclohexylcarbodiimide and dimethylaminopyridine. Wang resin orsimilar resins are used as support material for the syntheses.

The following derivatives of L-amino acids are used in the synthesis ofthe sequence, starting from the peptidyl resin as specified: a)hPTH(1-10) Seq. I.D. No. 1: Fmoc-Asn(Trt)-Wang resin, Fmoc-His(Trt)-OH,Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Boc-Ser(tBu)-OH; b)hPTH(9-18) Seq. I.D. No. 7): Fmoc-Met-Wang resin, Fmoc-Ser(tBu)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Boc-His(Trt)-OH; c)hPTH(24-37) Seq. I.D. No. 18: Fmoc-Leu-Wang resin, Fmoc-Ala-OH,Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-His(Trt)-OH,Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Leu-OH.

The synthesis may be carried out by in situ activation using2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or derivatives thereof, orbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate(BOP) or derivatives thereof in the presence of diisopropylethylamine orN-methylmorpholine and 1-hydroxybenzotriazole, using a four- to tenfoldexcess of Fmoc-L-amino acid during the coupling reactions inN,N-dimethylformamide, N,N-dimethylacetamide or N-methylpyrrolidone.Removal of the Fmoc groups is effected using 20% piperidine or 2%piperidine and 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) inN,N-dimethylformamide, N,N-dimethylacetamide or N-methylpyrrolidone.Following synthesis, the resins are washed with 2-propanol anddichloromethane and dried to constant weight in a high vacuum.

Removal from the support and deprotection are carried out by reactingthe peptidyl resin with trifluoroacetic acid containing 5% scavenger,water, ethanediol, phenol or thioanisole for 30-90 minutes at roomtemperature, filtrating, washing with trifluoroacetic acid, andsubsequently precipitating with tert-butyl methyl ether. The precipitateis lyophilized from aqueous solution.

EXAMPLE 2

Purification and Analysis

The raw products are purified by chromatography on a C18 reversed phasecolumn (10 μm, buffer A: 0.01 N HCl in water; buffer B: 20% isopropanol,30% methanol, 50% water, 0.01 N HCl; gradient: 10-80% within 60 minutes;detection at 230 nm).

The purity of the products is determined using mass spectrometry and C18reversed phase chromatography.

EXAMPLE 3

Coupling to Carrier Protein

Used as carrier proteins are hemocyanin, thyroglobulin, bovine serumalbumin, ovalbumin, or mouse serum albumin. Coupling is performedaccording to the carbodiimide method by way of the carboxyl groups ofthe peptides. The peptide is activated in aqueous solution by reactionwith 1-ethyl-3-(3-methylaminopropyl)carbodiimide hydrochloride for 5minutes. Coupling is effected by adding the activated peptide to anaqueous solution of the carrier. The molar ratio is 1 peptide 50 aminoacids of the carrier protein. The reaction takes 4 hours.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 36                                            - (2) INFORMATION FOR SEQ ID NO: 1:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:                                   - Ser Val Ser Glu Ile Gln Leu Met His Asn                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 2:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                    - Ser Val Ser Glu Ile Gln Leu Met His                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 3:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                    - Ser Val Ser Glu Ile Gln Leu Met                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 4:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                    - Ser Val Ser Glu Ile Gln Leu                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 5:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                    - Ser Val Ser Glu Ile Gln                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 6:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                    - Ser Val Ser Glu Ile                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 7:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                    - His Asn Leu Gly Lys His Leu Asn Ser Met                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 8:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                    - Asn Leu Gly Lys His Leu Asn Ser Met                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 9:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                    - Leu Gly Lys His Leu Asn Ser Met                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 10:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                   - Gly Lys His Leu Asn Ser Met                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 11:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                   - Lys His Leu Asn Ser Met                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 12:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                   - His Leu Asn Ser Met                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 13:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                   - His Asn Leu Gly Lys His Leu Asn Ser                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 14:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                   - His Asn Leu Gly Lys His Leu Asn                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 15:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                   - His Asn Leu Gly Lys His Leu                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 16:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                   - His Asn Leu Gly Lys His                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 17:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                   - His Asn Leu Gly Lys                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 18:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                   - Leu Arg Lys Lys Leu Gln Asp Val His Asn Ph - #e Val Ala Leu                 #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 19:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                   - Arg Lys Lys Leu Gln Asp Val His Asn Phe Va - #l Ala Leu                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                   - Lys Lys Leu Gln Asp Val His Asn Phe Val Al - #a Leu                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                   - Lys Leu Gln Asp Val His Asn Phe Val Ala Le - #u                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                   - Leu Gln Asp Val His Asn Phe Val Ala Leu                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                   - Gln Asp Val His Asn Phe Val Ala Leu                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                   - Asp Val His Asn Phe Val Ala Leu                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                   - Val His Asn Phe Val Ala Leu                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:                                   - His Asn Phe Val Ala Leu                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:                                   - Asn Phe Val Ala Leu                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 28:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:                                   - Leu Arg Lys Lys Leu Gln Asp Val His Asn Ph - #e Val Ala                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 29:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:                                   - Leu Arg Lys Lys Leu Gln Asp Val His Asn Ph - #e Val                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 30:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:                                   - Leu Arg Lys Lys Leu Gln Asp Val His Asn Ph - #e                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 31:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:                                   - Leu Arg Lys Lys Leu Gln Asp Val His Asn                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO: 32:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:                                   - Leu Arg Lys Lys Leu Gln Asp Val His                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 33:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:                                   - Leu Arg Lys Lys Leu Gln Asp Val                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 34:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:                                   - Leu Arg Lys Lys Leu Gln Asp                                                 1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 35:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                   - Leu Arg Lys Lys Leu Gln                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO: 36:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                               -     (ii) MOLECULE TYPE: peptide                                             -    (iii) HYPOTHETICAL: no                                                   -     (iv) ANTI-SENSE: no                                                     - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                   - Leu Arg Lys Lys Leu                                                         1               5                                                             __________________________________________________________________________

We claim:
 1. A kit for detecting active human parathyroid hormone (hPTH)comprising a container and a first group of antibodies or antibodyfragments and a second group of antibodies or antibody fragments,wherein the first group selectively binds a peptide of hPTH selectedfrom the group consisting of peptides having SEQ. ID. Nos. 1-6 and thesecond group selectively binds hPTH at an epitope contained within aminoacids 24 to
 37. 2. The kit of claim 1, wherein the second group ofantibodies or antibody fragments selectively binds a peptide of hPTHselected from the group consisting of peptides having SEQ. ID. Nos.18-36.
 3. The kit of claim 1, wherein the first group of antibodies orantibody fragments selectively bind peptides of hPTH having SEQ. ID.No.
 1. 4. The kit of claim 1, wherein the first group of antibodies orantibody fragments selectively bind peptides of hPTH having SEQ. ID. No.2.
 5. The kit of claim 1, wherein the first group of antibodies orantibody fragments selectively bind peptides of hPTH having SEQ. ID. No.3.
 6. The kit of claim 1, wherein the first group of antibodies orantibody fragments selectively bind peptides of hPTH having SEQ. ID. No.4.
 7. The kit of claim 1, wherein the first group of antibodies orantibody fragments selectively bind peptides of hPTH having SEQ. ID. No.5.
 8. The kit of claim 1, wherein the first group of antibodies orantibody fragments selectively bind peptides of hPTH having SEQ. ID. No.6.
 9. An immunological method of detecting active human parathyroidhormone (hPTH) in a sample comprising:contacting the sample with a firstantibody or antibody fragment which selectively binds a peptide of hPTHselected from the group consisting of peptides having SEQ. ID. Nos. 1-6,wherein the first antibody or antibody fragment binds hPTH in thesample; contacting the sample with a second antibody or antibodyfragment which selectively binds hPTH at an epitope contained withinamino acids 24 to 37; wherein the second antibody or antibody fragmentbinds to hPTH bound by the first antibody or antibody fragment; anddetecting the binding of the first and second antibodies or antibodyfragments, wherein the binding of the first and second antibodies orantibody fragments indicates the presence of active hPTH in the sample.10. The method of claim 9, wherein the second antibody or antibodyfragment selectively binds a peptide of hPTH selected from the groupconsisting of peptides having SEQ. ID. Nos. 18-36.
 11. The method ofclaim 9, wherein the first antibody or antibody fragment selectivelybinds peptides of hPTH having SEQ. ID. No.
 1. 12. The method of claim 9,wherein the first antibody or antibody fragment selectively bindspeptides of hPTH having SEQ. ID. No.
 2. 13. The method of claim 9,wherein the first antibody or antibody fragment selectively bindspeptides of hPTH having SEQ. ID. No.
 3. 14. The method of claim 9,wherein the first antibody or antibody fragment selectively bindspeptides of hPTH having SEQ. ID. No.
 4. 15. The method of claim 9,wherein the first antibody or antibody fragment selectively bindspeptides of hPTH having SEQ. ID. No.
 5. 16. The method of claim 9,wherein the first antibody or antibody fragment selectively bindspeptides of hPTH having SEQ. ID. No.
 6. 17. A composition comprising anantibody or antibody fragment and a suitable carrier, wherein theantibody or antibody fragment selectively binds a peptide of humanparathyroid hormone (hPTH) selected from the group consisting ofpeptides having SEQ. ID. Nos. 1-6.
 18. The composition of claim 17,wherein the composition further comprises a second antibody or antibodyfragment, wherein the second antibody or antibody fragment selectivelybinds hPTH at an epitope contained within amino acids 24 to
 37. 19. Thecomposition of claim 17, wherein the second antibody or antibodyfragment selectively binds a peptide of hPTH selected from the groupconsisting of peptides having SEQ. ID. Nos. 18-36.
 20. The compositionof claim 17, wherein the antibody or antibody fragment selectively bindspeptides of hPTH having SEQ. ID. No.
 1. 21. The composition of claim 17,wherein the antibody or antibody fragment selectively binds peptides ofhPTH having SEQ. ID. No.
 2. 22. The composition of claim 17, wherein theantibody or antibody fragment selectively binds peptides of hPTH havingSEQ. ID. No.
 3. 23. The composition of claim 17, wherein the antibody orantibody fragment selectively binds peptides of hPTH having SEQ. ID. No.4.
 24. The composition of claim 17, wherein the antibody or antibodyfragment selectively binds peptides pf hPTH having SEQ. ID. No.
 5. 25.The composition of claim 17, wherein the antibody or antibody fragmentselectivley binds peptides of hPTH having SEQ. ID. No. 6.